PROJECT SUMMARY Our long-term goal is to understand the function of nerves within bone, how changes to skeletal innervation with disease impact bone pathophysiology, and whether bioelectric stimulation can be used to promote skeletal health and bone accrual. Despite decades of research, there is only one clinical anabolic therapy to treat patients with bone loss and elevated fracture risk - and its use is limited to a total of only 2-years during a patient?s lifetime. Bioelectric stimulation of nerve outflow to skeletal tissues represents a novel, untapped option with significant clinical potential. Current literature supports a paradigm by which sensory neurotransmitters are anabolic, promoting bone formation, while the sympathetic neurotransmitter norepinephrine induces bone loss. Thus, selective activation of sensory nerve depolarization and release of efferent neurotransmitters may promote bone formation and contribute to decreased skeletal fragility in high- risk populations. A substantial barrier to taking such a targeted approach is our limited understanding of nerve distribution and function within the bone and bone marrow. To overcome this, the work in this proposal will establish critical fundamental insights into three key aspects of skeletal neurobiology. In Aim 1, we will define the connection and overlap of multiple, diverse skeletal regions across the central neuroaxis, including sub- classification of traced first-order sensory neurons in the dorsal root and trigeminal ganglia. In Aim 2, we will quantify the distribution and density of sensory and sympathetic nerves within the bone and bone marrow, and its variability across skeletal sites in mice and humans. This analysis will be paired with characterization of functional secondary architecture including the presence of Schwann cells around skeletal axons and terminal target relations with bone-forming osteoblasts, bone-resorbing osteoclasts, and hematopoietic cells. In Aim 3, we will directly examine the impact of bioelectric stimulation on bone and bone marrow including acute changes in soluble signaling factors and chronic regulation of skeletal health and integrity. The fundamental knowledge developed from the work in this proposal will provide essential information about the distribution and function of skeletal nerves to the SPARC consortium and inform future targeted approaches for bioelectric stimulation of skeletal health and regeneration.